Hello.

I want to talk about band forming experiments in the AUC.

That's a topic which is pretty old.

Because some of you may not know what band forming is, I will give you a short introduction.

In this video, we will speak about the dynamic gradient, which is the main problem.

Why it's probably not known by you by now.

And then about the analysis method and at the end a summary.

The key part of the band forming experiment is a slightly modified centerpiece with such

a reservoir here.

Where you can fill in liquid which gets then overlaid on top of the sector when the rotor

is accelerating.

For that you need a little density difference between the phases.

But 0.006 gram per milliliter is already enough to make the overlay properly working.

And the main difference in the data is then how the data looks.

Instead of an integrated concentration distribution over the radius, you get like a differential

one where you have those bands and they are quite broadened during the time by diffusion

while the middle of it is sedimenting down.

The first publication of this was 1963 by Vinograd and he already mentioned a few benefits

of band forming experiments.

Like the increased physical separation, the differential concentration distribution.

Back then it was only available with the Stieren optic later then.

But at the point of publishing this, there was only sedimentation velocity known.

So no differential distribution and it was very beneficial to the time and the technique

of recording data then with photo paper.

Then also less sample is needed.

It has to be a little bit more concentrated in the reservoir.

But overall you need less by the factor of between 4 and 10 depending on what you're

exactly measuring and how good your detector is.

And at the end you can use it for samples that are not purified.

They can be highly unpurified and with other components of your synthesis.

The little bit downside of it is it is more complex in an initial condition.

It's optically challenging because we have at the beginning this very concentrated band

and at the end a very broad band with very low concentration.

And then we have often non-ideal effects and due to the density difference needed, we need

some sort of density difference and therefore a little bit restricted in the solvents we

can use.

So how does it look and why do you want to know about it?

When the computer era arrives for AUC, we started fitting our data and we really soon

noticed that it doesn't work that well if you do a band forming experiment and want

to fit it.

This is due to a dynamic gradient of viscosity and density after the overlay because remember

we have two liquids with different densities and often in case of water and heavy water,

they also have different viscosities and they don't stay as completely separated phases

after the overlay, they diffuse into each other.

What you see down here is one microliter overlay of water onto heavy water for myoglobin and

the attempt of analyzing it with the given method of ZFIT or for example Ultrascan and

you can clearly see that especially the first few peaks are fitted pretty poorly and that

affects our result.

There were attempts of modeling this gradient with the second fixed flaw and some boundary

conditions but at the end it doesn't work that well that it could be used.